- Email: [email protected]
Soil moisture affects the interaction between Gaeumannomyces graminis var. Tritici and antagonistic bacteria
Soil Eiol. Biochem. Vol. 17, No. 4, pp. 44146, Printed in Great Britain. All rights reserved
1985 Copyright
0
003%0717/85 $3.00 + 0.00 1985 Pergamon Press Ltd
SOIL MOISTURE AFFECTS THE INTERACTION BETWEEN GAEUMANNOMYCES GRAMINIS VAR. TRITICI AND ANTAGONISTIC BACTERIA R. CAMPBELL and A. CLOR Department of Botany, University of Bristol, Bristol BS8 lUG, England (Accepted 10 January 1985) Summary-Reduction
in the soil moisture from a potential of - 10 to - 108 kPa reduced the growth of Bacillus cereus only spread through the soil or sand at potentials between - 10 and -60 kPa. The spread of B. pumilus was not affected by the water potential within the range tested. Both B. cereus and B. pumilus were less antagonistic in the drier sand and soil. When the bacteria and the fungus were grown together the maximum growth of the fungus occurred at those potentials when bacterial antagonism was reduced but the fungus was still able to grow. Gaeumannomyces
graminis. The antagonist
INTRODUCTION Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker causes a root rot of wheat called take-all. There have been many reports of microorganisms antagonistic to the fungus (Cook and Baker, 1983) and some of these may be giving biological control of the disease in take-all decline or suppressive soils. Interactions between the pathogen and the antagonists may be affected by many factors including the clay content of the soil and the water potential (Campbell, 1983; Campbell and Ephgrave, 1983). Different microorganisms will tolerate different degrees of water stress: in general, plant pathogenic fungi grow at low potentials (- 100 to - 1500 kPa) though they will grow slowly or survive down to - 5000 or - 10000 kPa (Parr et al., 1981; Cook and Baker, 1983). There is, however, great variation between species, and some are known to be favoured by wet soil conditions and others are -most active in drought-affected soils. The response of the host may be different to that of the pathogen. G. graminis var. tritici grows best in wet soils (matric potential - 100 kPa or less) though it will tolerate potentials down to - 5000 kPa, growth under these conditions is very slow or absent (Cook et al., 1972). The most obvious symptoms of the disease are, however, shown under dry conditions when the loss of roots causes the host to be most water stressed. Bacteria have been classified into groups based on their ability to tolerate dry conditions (Parr et al., 198 1): Bacillus and many other Gram-positive bacteria tolerate -2500 kPa though they may only grow in much wetter soils, above - 100 kPa. In considering the likely efficiency of biological control measures it is therefore essential to consider the response of the pathogen and the potential bacterial antagonists to soil water potential, the main component of which is the matric potential in most soils. Our aim was to determine the growth response of G. graminis var. tritici and the growth or movement through the soil of the particular antagonistic
bacteria that have been used as potential agents against it.
biocontrol
MATERIALS AND METHODS Gaeumannomyces graminis var. tritici was isolated from infected wheat roots and kept in the stock culture collection of the Department of Botany, University of Bristol on autoclaved whole wheat grains. Cultures were grown on malt agar (Oxoid) at 20°C and uniform discs cut from the colony margins with a sterile cork borer for use as standard inocula in all experiments. Two bacterial antagonists to G. graminis, Bacillus cereus var. mycoides, and B. pumilus, were isolated from wheat roots and kept as freeze-dried cultures in the Department of Botany culture collection. They were inoculated onto tryptic soy agar (Difco 0370-17), used at l/l0 the recommended strength, and then grown in l/l0 strength tryptic soy broth at 20°C for 4 days to give suspensions containing approximately 1O8 bacteria ml - ‘. The fungus and the bacteria were grown either singly or together in plates of sand or soil. These were glass Petri dishes partly filled with a mixture of sand and ground wheat straw or soil and ground wheat straw (500 g dry sand or soil plus 15 g ground wheat straw). The soil was a sandy loam from Long Ashton Research Station, University of Bristol, and had been used for wheat for 2 yr, preceded by grass. The soil for all the experiments was collected at one time, air dried and sieved (~2 mm) before use. It had the following composition by weight: 220.6 mm (coarse sand) 18.3x, 0.6-0.212mm (medium sand) 26.3x, 0.212-0.063 mm (fine sand) 46.8x, co.063 mm (silt and clay) 8.6%. This latter fraction had the following composition: 20-60pm (coarse silt) l.O%, 2&6pm (medium silt) 4.0x, 6-2pm (fine silt) 2.5%, ~2 pm (clay) 1.1%. The pH of the soil, in water, was 7.4. The sand was sea sand, well washed and then air dried before use. It had the following particle size distribution by weight: >2 mm 0.6%, 2-0.5 mm 441
442
R.
CAMPBELL and
13.1’i;, 0.5-0.25 mm 72.5%, 0.25-0.125 mm 10.4x, co.125 mm 0.2;/,. The control of the water potential was achieved as follows. A pressure plate was used to determine the relationship between the water potential of the soil or sand mixture and its water content (Fig. 1). The required water content for a given potential was read off the graph and water added to the dry sand or soil prior to autoclaving in covered jars. Extra water was also added to compensate for the water loss during autoclaving: this amount had been determined in previous experiments. The autoclaved sand or soil mixtures, now at the correct potential, were then emptied into sterile glass Petri dishes and pressed down with the base of a sterile flask. The fungus and the bacteria were inoculated into the edge of the sand or soil, on opposite sides if both were used, and were incubated at 20°C in closed boxes with high humidity. Preliminary experiments showed that this procedure gave plates with a reproducible water potential that did not alter significantly during incubation. The fungus grew across the dish, through the sand or soil mixture (Figs 5 and 7) and its growth was assessed by simple radial measurement. The bacterial spread from the point inoculum was assessed by Stotzky’s replica plate method (Stotzky, 1965). A sterile metal disc with closely set metal spikes was pressed into the sand or soil plate and then into a plate of sterile tryptic soy agar. The extent of the spread through the soil was found by recording where the colonies grew on the agar and relating this to the original plate. This assessment could be done repeatedly on each sand or soil plate to study the spread with time. The marks left by the metal spikes can be seen in Fig. 5. It was therefore possible to produce sterile sand or soil plates with wheat straw in them at given water potentials, and to study the growth or spread of the fungus and the bacteria through these mixtures when inoculated singly and in combination. RESULTS
The relationship between the sand and soil water contents and their matric potentials is as expected (Fig. l), the soil which contained clay and other colloidal material, required higher water contents for a given potential. The rates of growth, the final colony diameter of G. graminis and the spread of the antagonistic bacteria were similar in both sand and soil at a given potential. The results for sand will be presented in detail, since they cover a more complete range of potentials, and those from soil compared with them. The growth of G. graminis in the sand-wheat mixture decreases from wet to drier conditions, though at the greatest potential used (- 108 kPa) there is still some growth (Fig. 2). At day 19 the colony radii at potentials of - 10, - 20 and - 50 kPa were not different from each other, but they were all different from the growth at other potentials. Growth a -50 kPa was not different from -37 kPa, but all other possible comparisons were significantly different mostly at P < 0.01. In soil G. graminis growth showed a similar pattern with a decrease in growth rate down to - 80 kPa, the
A. CLOR
‘\‘\ SOll
.
Water potential I- kPa)
Fig. 1. The relationship between the water potential (- kPa) and the water content (% w/w) of the sand-straw and the soil-straw mixtures.
driest potential tested, though it grew rather more slowly in soil than in sand at the same potential. The results with B. cereus are summarized in Fig. 3 which is a combination of two separate experiments so all possible comparisons cannot be made, but it is clear that on sand-wheat straw mixtures wetter than -60 kPa, the growth or spread of B. cereus not affected by moisture content. At potentials greater than -90 kPa the bacterium did not grow or spread across the plate. The values at -90 and - 108 are significantly different from the others (P < 0.01) but not from each other. In soil B. cereus showed a more steady decrease in spread rather than the clustered values for sand and there was not the sudden decrease between - 60 and -90 kPa. B. pumilus spread very rapidly over the plates, covering them in less than 2 days at all potentials used in both sand and soil mixtures. These data for the fungus and the bacterium growing alone explain the results obtained when both organisms were inoculated onto opposite sides of the same plate (Fig. 4 for B. cereus). Considering the results in general, the fungus still grows best in wetter conditions, though bacterial antagonism reduces its growth when the colonies meet (Fig. 5; cf. Figs 4 and 2). In drier conditions however (-90 and - 108 kPa) the inhibition is removed (Fig. 3) so the fungus grows comparatively well (Fig. 5). There was not a steady fall-off in growth as the sand-wheat straw became drier since at - 90 kPa the fungus is still able to grow quite well, but the bacterium not at all. So by day 28 the uninhibited growth of the fungus at -90 kPa is better than the reduced growth at -60 (P < 0.05) and -50 kPa (P < 0.01). Growth at - 108 kPa is worse than at - 90 because of the water effect on the fungus, but even this poor growth rate eventually gives a greater colony diameter than wetter potentials because the fungus is not inhibited by the bacteria. The driest condition (-80 kPa) in soils gave the maximum radial growth of G. graminis after 25 days as the fungus was not inhibited and again in the wetter soils growth started quickly but was then
443
Soil water and microbial antagonism
6E " 5!: .0 e
4-
JEr 0 L m
3-
I
I
I
I
I
I
I
I
7
9
12
14
19
21
23
26
Incubation
Fig. 2. The effect of water
potential
(from
(days
1
- 10 to - 108 kPa) on growth straw plates.
inhibited by the activity B. cereus and so finished with a lesser diameter than the uninhibited, slower growing fungus in the drier plates, which was not affected by the bacterium. The reaction with B. pumilus in sand is an extreme example of that just described. The bacterium grew across the plates and completely inhibited growth of G. graminis at potentials wetter than -90 kPa (Fig. 7). At -90 and - 108 kPa the fungus grew slightly (Fig. 6) but much less than without the bacterium (cf. Figs 6 and 2). Even though B. pumilus spreads across the plates under these drier conditions it is apparently less inhibitory than in wetter mixtures of sand. In soil B. pumilus caused no inhibition of growth of G. graminis at -80 kPa though it was completely inhibitory in wetter soils. DISCUSSION
Grose et al. (1984) have studied the growth of G. temperatures and at two graminis at different different water levels in each of two soils. They used water potentials of -0.2 to - 10 kPa which is much wetter than the soil or sand used in our study even though they considered this equivalent to a waterholding capacity of 3&60x and called their levels “wet” and “moist”. They postulated that in wetter soils which were unsterilized, the reduction in growth of G. graminis which they observed could be due to antagonists, though there was no direct evidence of what these might be or any measure of their activity. However our work suggests that the hypothesis could be correct since the two known antagonists used did only operate in wetter conditions, though all the water levels used were less than those of Grose et al. (1984). The increased growth of G. graminis in wetter soils is in agreement with Cook et al. (1972) though they showed growth right down to -4800 kPa. Similarly
I 26
of G. graminis
in sand-wheat
our study confirmed the findings of Peterson et al. (1965) that Bacillk grows best at high soil moisture levels. B. cereus grew down to -90 kPa, but other bacteria may stop in much wetter conditions: Pseudo monas has been reported to grow only above -20 kPa (Bowen, 1979). Even though spread across the plate occurs in relatively dry conditions there was evidence, especially from B. pumilus, that the ability of this bacterium to antagonize G. graminis decreases as the water potential is increased, before spread is severely inhibited. The result of the interaction between this strain of G. graminis and B. cereus and B. pumilus is dependent on the water potential in the gnotobiotic conditions used. The situation in the field, where the bacteria have been shown to give some control of the take-all disease caused by G. graminis (Campbell and Faull, 1979), will be much more complex. Our study suggests however, that the biocontrol agents may only give control under wetter conditions and that there
1
5
I 7
I
I
1
I
I
IO
12
14
16
19
Incubation
I 21
(days)
Fig. 3. The effect of water potential (- 10 to - 108 kPa) on the spread of B. cereu~ across sand-wheat straw plates.
R.
I 7
CAMPBELL
and A.
CLOR
I
I
L
I
I
i
I
I
1
9
12
14
16
19
21
23
26
28
incubation
(days)
Fig. 4. The effect of water potential (from - 10 to - 108 kPa) on the growth of G. grami& in sand--wheat straw plates in the presence of B. cereus.
Fig. 5. The growth of G. gmminis in soil and wheat straw, with (bottom) and without (top) B. cereus. There are two different potentials, - 10 kPa (left) and -80 kPa (right). Inhibition is greatest at - 10 kPa where the bacteria are active. Note the marks made by the metal replicater in the bottom dishes.
445
Soil water and microbial antagonism
s .0 z
3-
i
2-
e m c" 0 z v
A-A-108 d
O-90
.
Gl-
.-
;_./AA
I 7
I 9
, 11
I 13
Incubation
I 15
1 17
I 19
1 21
I 24
(days)
Fig. 6. The effect of water potential (at -90 and - 108 kPa) on the growth of G. graminis in sand-wheat straw plates in the presence of B. pun&s. There was no growth, because of inhibition of the fungus by the bacterium, at any potentials wetter than -90 kPa, so these zero values are not shown.
Fig. 7. The effect of B. pumilus on growth of G. gruminis in sand-wheat straw at - 50 kPa: there is almost complete inhibition. The bacterium was inoculated at the bottom of the right hand plate, and the fungus at the top of both plates.
could be situations where the fungus could grow in drier soils when the bacteria were no longer antagonistic. In addition the field situation is complicated by the response of the wheat plant. Though infection may be greater in wet soils, the effect of the disease may be worse when the plant is drought stressed, since infected plants have fewer viable roots. Thus the bacteria may be able to grow under wet conditions when there is the greatest problem with infection and spread of G. graminis but may be less effective when drought stress results in severe symptoms on the plant. Cassell and Hering (1982) showed that, in soil, G. graminis infection was not changed by potentials between - 100 and - 10 kPa but the disease was more severe in the wetter soil. A further complication in natural soils is that, apart from the water content per se, the matric potential will also be affected by the particle size and hence the clay content of the soil. There are also effects of clay on the bacteria independent of water potential (Campbell and Ephgrave, 1983).
These basic studies on the interactions of the fungus with two bacteria need to be extended to more natural agricultural situations where many other factors will be interacting, but they confirm that such microbial interactions may be extremely complex and that environmental factors may well limit the effectiveness of potential biocontrol agents.
REFERENCES Bowen G. D. (1979) Integrated and experimental approaches to the study of growth of organisms around roots. In Soil-Borne Plant Pathogens (B. Schippers and W. Gams, Eds), pp. 209-227. Academic Press, London. Campbell R. (1983) Ultrastructural studies of Gaeumannomyces gruminis in the water films on wheat roots and the effect of clay on the interaction between this fungus and antagonistic bacteria. Canadian Journal of Microbiology 29, 3945. Campbell R. and Ephgrave J. M. E. (1983) Effect of bentonite clay on the growth of Gaeumannomyces gram-
446
R. CAMPBELLand A. CLOR
&is var. tritici and on its interactions with antagonistic bacteria. ~~~rn~~ of Gemw.zl ~jcrob~o~o~~ 129. 771-777. Campbell R. and F&l J. L. (1979) Biological control of Gaeumunnoq~ce.~ gr~tizirris: held trials and the uitrastructure of the interactions with antagonistic bacteria. In Soil-Borne Plant Pathogens (B. Schippers and W. Gams, Eds), pp. 603%610. Academic Press, London. Cassell D. and Hering T. F. (1982) The effect of water potential on soil-borne diseases of wheat seedlings. Annuls of Appiied Biologv 101, 367-375. Cook R. J. and Baker K. F. (1983) The Natwe and Practice qf Biologjeul Conrro/ of‘ Plum ~at~loge~z.~.American Phytopathological Society, St. Paul, Minnesota. Cook R. J., Pappendick R. 1. and Griffin D. M. (1972) Growth of two root-rot fungi as affected by osmotic and
manic water potentials. Soil Science Socieq of America Pr~~cee~~ing.~36, 78-82. Grose M. J., Parker C. A. and Sivasithamparam K. (1984j Growth of Gaeumannomyces grumittis var. tritici in soil: effects of temperature and water potential. Svil Biology & BiochemktrJ 16, 21 I-216. Parr J. F., Gardner W. R. and Elliot L. F. (Eds) (1981) Wuter Potential Relations in Soil Microbiology. Soil Science Society of America, Madison, Wisconsin. Peterson E. A., Rouatt J. W. and Katznelson H. (1965) .~icroorganisms in the root zone in relation to soil moisture. Cunudian Journal qf microbiology 11, 483495. Stotzky G. (1965) Replica plating technique for studying microbial interactions in soil. Cannclian Journal of MicrohiologF 11, 629636.
1985 Copyright
0
003%0717/85 $3.00 + 0.00 1985 Pergamon Press Ltd
SOIL MOISTURE AFFECTS THE INTERACTION BETWEEN GAEUMANNOMYCES GRAMINIS VAR. TRITICI AND ANTAGONISTIC BACTERIA R. CAMPBELL and A. CLOR Department of Botany, University of Bristol, Bristol BS8 lUG, England (Accepted 10 January 1985) Summary-Reduction
in the soil moisture from a potential of - 10 to - 108 kPa reduced the growth of Bacillus cereus only spread through the soil or sand at potentials between - 10 and -60 kPa. The spread of B. pumilus was not affected by the water potential within the range tested. Both B. cereus and B. pumilus were less antagonistic in the drier sand and soil. When the bacteria and the fungus were grown together the maximum growth of the fungus occurred at those potentials when bacterial antagonism was reduced but the fungus was still able to grow. Gaeumannomyces
graminis. The antagonist
INTRODUCTION Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker causes a root rot of wheat called take-all. There have been many reports of microorganisms antagonistic to the fungus (Cook and Baker, 1983) and some of these may be giving biological control of the disease in take-all decline or suppressive soils. Interactions between the pathogen and the antagonists may be affected by many factors including the clay content of the soil and the water potential (Campbell, 1983; Campbell and Ephgrave, 1983). Different microorganisms will tolerate different degrees of water stress: in general, plant pathogenic fungi grow at low potentials (- 100 to - 1500 kPa) though they will grow slowly or survive down to - 5000 or - 10000 kPa (Parr et al., 1981; Cook and Baker, 1983). There is, however, great variation between species, and some are known to be favoured by wet soil conditions and others are -most active in drought-affected soils. The response of the host may be different to that of the pathogen. G. graminis var. tritici grows best in wet soils (matric potential - 100 kPa or less) though it will tolerate potentials down to - 5000 kPa, growth under these conditions is very slow or absent (Cook et al., 1972). The most obvious symptoms of the disease are, however, shown under dry conditions when the loss of roots causes the host to be most water stressed. Bacteria have been classified into groups based on their ability to tolerate dry conditions (Parr et al., 198 1): Bacillus and many other Gram-positive bacteria tolerate -2500 kPa though they may only grow in much wetter soils, above - 100 kPa. In considering the likely efficiency of biological control measures it is therefore essential to consider the response of the pathogen and the potential bacterial antagonists to soil water potential, the main component of which is the matric potential in most soils. Our aim was to determine the growth response of G. graminis var. tritici and the growth or movement through the soil of the particular antagonistic
bacteria that have been used as potential agents against it.
biocontrol
MATERIALS AND METHODS Gaeumannomyces graminis var. tritici was isolated from infected wheat roots and kept in the stock culture collection of the Department of Botany, University of Bristol on autoclaved whole wheat grains. Cultures were grown on malt agar (Oxoid) at 20°C and uniform discs cut from the colony margins with a sterile cork borer for use as standard inocula in all experiments. Two bacterial antagonists to G. graminis, Bacillus cereus var. mycoides, and B. pumilus, were isolated from wheat roots and kept as freeze-dried cultures in the Department of Botany culture collection. They were inoculated onto tryptic soy agar (Difco 0370-17), used at l/l0 the recommended strength, and then grown in l/l0 strength tryptic soy broth at 20°C for 4 days to give suspensions containing approximately 1O8 bacteria ml - ‘. The fungus and the bacteria were grown either singly or together in plates of sand or soil. These were glass Petri dishes partly filled with a mixture of sand and ground wheat straw or soil and ground wheat straw (500 g dry sand or soil plus 15 g ground wheat straw). The soil was a sandy loam from Long Ashton Research Station, University of Bristol, and had been used for wheat for 2 yr, preceded by grass. The soil for all the experiments was collected at one time, air dried and sieved (~2 mm) before use. It had the following composition by weight: 220.6 mm (coarse sand) 18.3x, 0.6-0.212mm (medium sand) 26.3x, 0.212-0.063 mm (fine sand) 46.8x, co.063 mm (silt and clay) 8.6%. This latter fraction had the following composition: 20-60pm (coarse silt) l.O%, 2&6pm (medium silt) 4.0x, 6-2pm (fine silt) 2.5%, ~2 pm (clay) 1.1%. The pH of the soil, in water, was 7.4. The sand was sea sand, well washed and then air dried before use. It had the following particle size distribution by weight: >2 mm 0.6%, 2-0.5 mm 441
442
R.
CAMPBELL and
13.1’i;, 0.5-0.25 mm 72.5%, 0.25-0.125 mm 10.4x, co.125 mm 0.2;/,. The control of the water potential was achieved as follows. A pressure plate was used to determine the relationship between the water potential of the soil or sand mixture and its water content (Fig. 1). The required water content for a given potential was read off the graph and water added to the dry sand or soil prior to autoclaving in covered jars. Extra water was also added to compensate for the water loss during autoclaving: this amount had been determined in previous experiments. The autoclaved sand or soil mixtures, now at the correct potential, were then emptied into sterile glass Petri dishes and pressed down with the base of a sterile flask. The fungus and the bacteria were inoculated into the edge of the sand or soil, on opposite sides if both were used, and were incubated at 20°C in closed boxes with high humidity. Preliminary experiments showed that this procedure gave plates with a reproducible water potential that did not alter significantly during incubation. The fungus grew across the dish, through the sand or soil mixture (Figs 5 and 7) and its growth was assessed by simple radial measurement. The bacterial spread from the point inoculum was assessed by Stotzky’s replica plate method (Stotzky, 1965). A sterile metal disc with closely set metal spikes was pressed into the sand or soil plate and then into a plate of sterile tryptic soy agar. The extent of the spread through the soil was found by recording where the colonies grew on the agar and relating this to the original plate. This assessment could be done repeatedly on each sand or soil plate to study the spread with time. The marks left by the metal spikes can be seen in Fig. 5. It was therefore possible to produce sterile sand or soil plates with wheat straw in them at given water potentials, and to study the growth or spread of the fungus and the bacteria through these mixtures when inoculated singly and in combination. RESULTS
The relationship between the sand and soil water contents and their matric potentials is as expected (Fig. l), the soil which contained clay and other colloidal material, required higher water contents for a given potential. The rates of growth, the final colony diameter of G. graminis and the spread of the antagonistic bacteria were similar in both sand and soil at a given potential. The results for sand will be presented in detail, since they cover a more complete range of potentials, and those from soil compared with them. The growth of G. graminis in the sand-wheat mixture decreases from wet to drier conditions, though at the greatest potential used (- 108 kPa) there is still some growth (Fig. 2). At day 19 the colony radii at potentials of - 10, - 20 and - 50 kPa were not different from each other, but they were all different from the growth at other potentials. Growth a -50 kPa was not different from -37 kPa, but all other possible comparisons were significantly different mostly at P < 0.01. In soil G. graminis growth showed a similar pattern with a decrease in growth rate down to - 80 kPa, the
A. CLOR
‘\‘\ SOll
.
Water potential I- kPa)
Fig. 1. The relationship between the water potential (- kPa) and the water content (% w/w) of the sand-straw and the soil-straw mixtures.
driest potential tested, though it grew rather more slowly in soil than in sand at the same potential. The results with B. cereus are summarized in Fig. 3 which is a combination of two separate experiments so all possible comparisons cannot be made, but it is clear that on sand-wheat straw mixtures wetter than -60 kPa, the growth or spread of B. cereus not affected by moisture content. At potentials greater than -90 kPa the bacterium did not grow or spread across the plate. The values at -90 and - 108 are significantly different from the others (P < 0.01) but not from each other. In soil B. cereus showed a more steady decrease in spread rather than the clustered values for sand and there was not the sudden decrease between - 60 and -90 kPa. B. pumilus spread very rapidly over the plates, covering them in less than 2 days at all potentials used in both sand and soil mixtures. These data for the fungus and the bacterium growing alone explain the results obtained when both organisms were inoculated onto opposite sides of the same plate (Fig. 4 for B. cereus). Considering the results in general, the fungus still grows best in wetter conditions, though bacterial antagonism reduces its growth when the colonies meet (Fig. 5; cf. Figs 4 and 2). In drier conditions however (-90 and - 108 kPa) the inhibition is removed (Fig. 3) so the fungus grows comparatively well (Fig. 5). There was not a steady fall-off in growth as the sand-wheat straw became drier since at - 90 kPa the fungus is still able to grow quite well, but the bacterium not at all. So by day 28 the uninhibited growth of the fungus at -90 kPa is better than the reduced growth at -60 (P < 0.05) and -50 kPa (P < 0.01). Growth at - 108 kPa is worse than at - 90 because of the water effect on the fungus, but even this poor growth rate eventually gives a greater colony diameter than wetter potentials because the fungus is not inhibited by the bacteria. The driest condition (-80 kPa) in soils gave the maximum radial growth of G. graminis after 25 days as the fungus was not inhibited and again in the wetter soils growth started quickly but was then
443
Soil water and microbial antagonism
6E " 5!: .0 e
4-
JEr 0 L m
3-
I
I
I
I
I
I
I
I
7
9
12
14
19
21
23
26
Incubation
Fig. 2. The effect of water
potential
(from
(days
1
- 10 to - 108 kPa) on growth straw plates.
inhibited by the activity B. cereus and so finished with a lesser diameter than the uninhibited, slower growing fungus in the drier plates, which was not affected by the bacterium. The reaction with B. pumilus in sand is an extreme example of that just described. The bacterium grew across the plates and completely inhibited growth of G. graminis at potentials wetter than -90 kPa (Fig. 7). At -90 and - 108 kPa the fungus grew slightly (Fig. 6) but much less than without the bacterium (cf. Figs 6 and 2). Even though B. pumilus spreads across the plates under these drier conditions it is apparently less inhibitory than in wetter mixtures of sand. In soil B. pumilus caused no inhibition of growth of G. graminis at -80 kPa though it was completely inhibitory in wetter soils. DISCUSSION
Grose et al. (1984) have studied the growth of G. temperatures and at two graminis at different different water levels in each of two soils. They used water potentials of -0.2 to - 10 kPa which is much wetter than the soil or sand used in our study even though they considered this equivalent to a waterholding capacity of 3&60x and called their levels “wet” and “moist”. They postulated that in wetter soils which were unsterilized, the reduction in growth of G. graminis which they observed could be due to antagonists, though there was no direct evidence of what these might be or any measure of their activity. However our work suggests that the hypothesis could be correct since the two known antagonists used did only operate in wetter conditions, though all the water levels used were less than those of Grose et al. (1984). The increased growth of G. graminis in wetter soils is in agreement with Cook et al. (1972) though they showed growth right down to -4800 kPa. Similarly
I 26
of G. graminis
in sand-wheat
our study confirmed the findings of Peterson et al. (1965) that Bacillk grows best at high soil moisture levels. B. cereus grew down to -90 kPa, but other bacteria may stop in much wetter conditions: Pseudo monas has been reported to grow only above -20 kPa (Bowen, 1979). Even though spread across the plate occurs in relatively dry conditions there was evidence, especially from B. pumilus, that the ability of this bacterium to antagonize G. graminis decreases as the water potential is increased, before spread is severely inhibited. The result of the interaction between this strain of G. graminis and B. cereus and B. pumilus is dependent on the water potential in the gnotobiotic conditions used. The situation in the field, where the bacteria have been shown to give some control of the take-all disease caused by G. graminis (Campbell and Faull, 1979), will be much more complex. Our study suggests however, that the biocontrol agents may only give control under wetter conditions and that there
1
5
I 7
I
I
1
I
I
IO
12
14
16
19
Incubation
I 21
(days)
Fig. 3. The effect of water potential (- 10 to - 108 kPa) on the spread of B. cereu~ across sand-wheat straw plates.
R.
I 7
CAMPBELL
and A.
CLOR
I
I
L
I
I
i
I
I
1
9
12
14
16
19
21
23
26
28
incubation
(days)
Fig. 4. The effect of water potential (from - 10 to - 108 kPa) on the growth of G. grami& in sand--wheat straw plates in the presence of B. cereus.
Fig. 5. The growth of G. gmminis in soil and wheat straw, with (bottom) and without (top) B. cereus. There are two different potentials, - 10 kPa (left) and -80 kPa (right). Inhibition is greatest at - 10 kPa where the bacteria are active. Note the marks made by the metal replicater in the bottom dishes.
445
Soil water and microbial antagonism
s .0 z
3-
i
2-
e m c" 0 z v
A-A-108 d
O-90
.
Gl-
.-
;_./AA
I 7
I 9
, 11
I 13
Incubation
I 15
1 17
I 19
1 21
I 24
(days)
Fig. 6. The effect of water potential (at -90 and - 108 kPa) on the growth of G. graminis in sand-wheat straw plates in the presence of B. pun&s. There was no growth, because of inhibition of the fungus by the bacterium, at any potentials wetter than -90 kPa, so these zero values are not shown.
Fig. 7. The effect of B. pumilus on growth of G. gruminis in sand-wheat straw at - 50 kPa: there is almost complete inhibition. The bacterium was inoculated at the bottom of the right hand plate, and the fungus at the top of both plates.
could be situations where the fungus could grow in drier soils when the bacteria were no longer antagonistic. In addition the field situation is complicated by the response of the wheat plant. Though infection may be greater in wet soils, the effect of the disease may be worse when the plant is drought stressed, since infected plants have fewer viable roots. Thus the bacteria may be able to grow under wet conditions when there is the greatest problem with infection and spread of G. graminis but may be less effective when drought stress results in severe symptoms on the plant. Cassell and Hering (1982) showed that, in soil, G. graminis infection was not changed by potentials between - 100 and - 10 kPa but the disease was more severe in the wetter soil. A further complication in natural soils is that, apart from the water content per se, the matric potential will also be affected by the particle size and hence the clay content of the soil. There are also effects of clay on the bacteria independent of water potential (Campbell and Ephgrave, 1983).
These basic studies on the interactions of the fungus with two bacteria need to be extended to more natural agricultural situations where many other factors will be interacting, but they confirm that such microbial interactions may be extremely complex and that environmental factors may well limit the effectiveness of potential biocontrol agents.
REFERENCES Bowen G. D. (1979) Integrated and experimental approaches to the study of growth of organisms around roots. In Soil-Borne Plant Pathogens (B. Schippers and W. Gams, Eds), pp. 209-227. Academic Press, London. Campbell R. (1983) Ultrastructural studies of Gaeumannomyces gruminis in the water films on wheat roots and the effect of clay on the interaction between this fungus and antagonistic bacteria. Canadian Journal of Microbiology 29, 3945. Campbell R. and Ephgrave J. M. E. (1983) Effect of bentonite clay on the growth of Gaeumannomyces gram-
446
R. CAMPBELLand A. CLOR
&is var. tritici and on its interactions with antagonistic bacteria. ~~~rn~~ of Gemw.zl ~jcrob~o~o~~ 129. 771-777. Campbell R. and F&l J. L. (1979) Biological control of Gaeumunnoq~ce.~ gr~tizirris: held trials and the uitrastructure of the interactions with antagonistic bacteria. In Soil-Borne Plant Pathogens (B. Schippers and W. Gams, Eds), pp. 603%610. Academic Press, London. Cassell D. and Hering T. F. (1982) The effect of water potential on soil-borne diseases of wheat seedlings. Annuls of Appiied Biologv 101, 367-375. Cook R. J. and Baker K. F. (1983) The Natwe and Practice qf Biologjeul Conrro/ of‘ Plum ~at~loge~z.~.American Phytopathological Society, St. Paul, Minnesota. Cook R. J., Pappendick R. 1. and Griffin D. M. (1972) Growth of two root-rot fungi as affected by osmotic and
manic water potentials. Soil Science Socieq of America Pr~~cee~~ing.~36, 78-82. Grose M. J., Parker C. A. and Sivasithamparam K. (1984j Growth of Gaeumannomyces grumittis var. tritici in soil: effects of temperature and water potential. Svil Biology & BiochemktrJ 16, 21 I-216. Parr J. F., Gardner W. R. and Elliot L. F. (Eds) (1981) Wuter Potential Relations in Soil Microbiology. Soil Science Society of America, Madison, Wisconsin. Peterson E. A., Rouatt J. W. and Katznelson H. (1965) .~icroorganisms in the root zone in relation to soil moisture. Cunudian Journal qf microbiology 11, 483495. Stotzky G. (1965) Replica plating technique for studying microbial interactions in soil. Cannclian Journal of MicrohiologF 11, 629636.